Antimicrobial composition having efficacy against endospores

ABSTRACT

A sporicidal composition has a moderately low pH and includes a glycosidase and the dissocia-tion product of at least one inorganic oxidizing agent. High effective solute concentrations can enhance the efficacy of the composition. The composition can be applied to a surface and allowed to absorb into an endospore, ultimately killing at least some of those bacteria in mature endospore form. Surfaces to be treated include inanimate (hard) surfaces via cleaning and medical devices such as endoscopes, typically via immersion.

CROSS REFERENCE TO RELATED APPLICATIONS

This international application claims the benefit of U.S. provisional patent application nos. 62/202,644 and 62/212,552, filed 7 Aug. 2015 and 31 Aug. 2015, respectively, the disclosures of which are incorporated herein by reference.

BACKGROUND INFORMATION

Unlike a true spore, an endospore is not an offspring of another living organism. Nevertheless, the terms “spore” and “endospore” are used interchangeably hereinthroughout.

A vegetative bacterium is one which can grow, feed and reproduce. When nutrients become scarce, certain vegetative bacteria begin a process referred to as “sporulation,” where they take on a reduced, dormant form which permits them to survive without nutrients and gives them resistance to UV radiation, desiccation, elevated temperatures, extreme freezing and chemical disinfectants. Soon after environmental conditions return to being favorable for vegetative growth, such bacteria can exit their dormant state (“spore germination”) with the spore core rehydrating, the cortex hydrolyzing, the coat being shed and, ultimately, DNA replication being initiated. For additional information on these complex processes, the interested reader is directed to any of a variety of texts such as, for example, J. C. Pommerville, Fundamentals of Microbiology, 10th ed. (Jones & Bartlett Learning; Burlington, Mass.).

At the outset of the sporulation step, a vegetative bacterium called a “mother cell” makes a copy of its DNA and then forms a membrane around the new copy of DNA, with the section of the cell having the copied DNA completely surrounded by a membrane being referred to as a “forespore.” The next morphological stage of sporulation is “engulfment” of the forespore by its mother cell, a process that is analogous to phagocytosis; when engulfment is complete, the forespore is entirely surrounded by its inner and outer membranes and free in the mother cell cytoplasm. Around this core of the spore is assembled a series of protective structures, completion of which results in a mature endospore, which is released after the mother cell is broken apart.

Endospores have a multi-layer structure, all of which protect the nucleus. The nucleus is protected by a number of layers, set forth below in Table 1 in order from inside-to-outside:

TABLE 1 Layer Description (1) core protoplast that contains RNA, DNA, dipicolinic acid, low molecular weight basic proteins, and various minerals such as Ca, K, Mn and P (2) cortical (outer) non-crosslinked or lightly crosslinked peptido- membrane glycan-containing layer that develops into cell wall during germination (3) cortex peptidoglycan and manuronic acid residues (4) inner spore coat primarily acidic polypeptides (5) outer spore coat primarily protein with some carbohydrates and lipids and, in the case of C. difficile, a large amount of phosphorus

The inner spore coat is soluble in alkali solutions, but the outer spore coat is resistant to hydrolysis from alkalis, probably due in significant part to its numerous disulfide (—S—S—) linkages.

Spores are extremely difficult to eradicate and are involved in the spread of diseases such as Clostridium difficile (C. diff) infection and anthrax.

C. difficile is a Gram-positive, spore-forming bacterium often found in healthcare facilities and is the cause of antibiotic-associated diarrhea. C. difficile infection is a growing problem, affecting hundreds of thousands of people each year, killing a significant portion of those affected. C. difficile spores are resistant to most routine surface cleaning methods, remaining viable in the environment for long periods of time.

Anthrax is an acute, usually lethal, disease that affects both humans and animals, caused by the bacterium Bacillus anthracis (B. anthracis), which spores can be produced in vitro and used as a biological weapon. Anthrax does not spread directly from one infected animal or person to another, instead being spread by spores.

Sporostatic compounds are not sporicidal, i.e., they do not kill spores; instead, they inhibit germination of spores or cause germinated spores to grow abnormally. Spores can survive exposure to these compounds and then grow after sporostatic compounds no longer are present. Sporostatic compounds include phenols and cresols, organic acids and esters, alcohols, quaternary ammonia compounds, biguanides, and organomercury compounds. Certain of these sporostatic compounds can be marginally sporicidal at high concentrations; see, e.g., A. D. Russell, “Bacterial Spores and Chemical Sporicidal Agents,” Clinical Microbiology Reviews, pp. 99-119 (1990)) for the relative concentrations of certain sporostatic compounds needed to achieve any sporicidal efficacy.

Commonly employed spore treatment options include aldehydes, particularly gluteraldehyde and formaldehyde; chlorine-releasing agents including Cl₂, sodium hypochlorite, calcium hypochlorite, and chlorine reducing agents such as dichloroisocyanurate; iodine and iodophors; peroxygens including hydrogen peroxide and peracetic acid; gases such as ethylene oxide, propylene oxide and ozone; and β-propiolactone. The mechanisms of their activities against spores are not particularly well understood although, in all cases, the activity is rate-limited by the permeation of the active molecule(s) through the protective layers of the spore. This need to penetrate the various layers of protection means that spores must be exposed to these products for long periods of time at high concentrations.

U.S. Pat. Nos. 8,940,792 and 9,314,017 as well as U.S. Pat. Publ. Nos. 2010/0086576, 2013/0272922, 2013/0079407 and 2016/0073628 describe antimicrobial compositions and various uses therefor. The core and cortex (including cortical membrane) of a spore are susceptible to dissolution and lysis by the types of high osmolarity compositions described in these documents. However, such compositions have not been found to be particularly effective against spores, likely due to a limited ability to break down and then penetrate the outer and inner spore coats.

That which remains desirable is a composition that is capable of penetrating the various defenses of endospores and killing bacteria therein. Such a composition preferably is effective against endospores of bacteria such as of C. difficile and B. anthracis while not presenting toxicity concerns toward humans who handle or contact it.

SUMMARY

In one aspect is provided a composition that can kill a variety of endospore-forming bacteria while in a mature endospore form. The composition is at least somewhat acidic, but its pH is at least 4.5. It includes a solute component that includes at least one acid, dissociation product of a buffer precursor, the dissociation product of at least one electrolyte oxidizing agent, and at least one glycosidase. In certain embodiments, the composition also can include one or more of an organic liquid, a wetting agent (particularly an ionic surfactant), and any of a variety of non-oxidizing electrolytes so as to increase the effective solute concentration of the composition. Advantageously, the composition need not include an active antimicrobial agent to be effectively sporicidal.

The composition includes a solvent component and a solute component that is present in an amount sufficient to result in an overall osmolarity of the composition of at least 0.5 Osm/L, generally at least 0.75 Osm/L and commonly at least 1 Osm/L, up to the solubility limit of the solute component in the solvent component.

In certain embodiments, the solvent component of the composition includes both water and organic liquid(s), with the solvent component exhibiting a dipolar intermolecular force Hansen Solubility Parameter (HSP) value of from 13.1 to 15.7 MPa^(1/2).

In these and other embodiments, the solute component includes a buffer that comprises at least one carboxylic acid and the dissociation product of a conjugate base of one of said at least one carboxylic acid, dissociation product of one or more electrolyte oxidizing agents, a glycosidase, and an ionic surfactant.

The composition advantageously is not strongly acidic, i.e., has a pH of 5.5±1.0, a range that is generally considered biocompatible; specifically, external exposure to some embodiments results in no long-term negative dermal effects and ingestion can result biodegradation and/or biosorption, particularly if diluted with water soon after ingestion.

The composition is effective at preventing endospore bacteria from returning to a vegetative state. Advantageously, in many embodiments, it is non-toxic or, at worst, has low toxicity. Many of the ingredients are generally considered to be biocompatible.

Embodiments of the composition are effective against the various defenses of bacteria in endospore form, specifically, disulfide bonds can be cleaved, intrinsic hydrophobicity can be overcome, peptidoglycan can be disrupted, and the core and cortical membrane can be lysed.

Also provided is a method of treating a surface. The method involves applying an embodiment of the foregoing composition to the surface and permitting the composition to be absorbed into the endospore and to kill at least some of those bacteria in mature endospore form. The surface being treated can be an inanimate surface, particularly a hard surface, and advantageously a hard surface in a healthcare facility. Advantageously, the composition can be used to treat medical devices, including but not limited to endoscopes, while having little to no negative effects on metal, glass, quartz or polymeric components thereto.

Embodiments of the composition can provide passing results in a test such as AOAC Official Method 966.04 within commercially relevant timeframes. For example, sterilizing treatment of medical instruments can be effected in less than 4 hours and, at times, no more than 30 minutes.

Other aspects of the invention will be apparent to the ordinarily skilled artisan from the detailed description that follows. To assist in understanding that description, certain definitions are provided immediately below, and these are intended to apply throughout unless the surrounding text explicitly indicates a contrary intention:

-   -   “comprising” means to include, but not be limited to, the listed         ingredients or steps;     -   “consisting of” means to include only the listed ingredients (or         steps) and minor amounts of inactive additives or adjuvants;     -   “consisting essentially of” means to include only the listed         ingredients (or steps), minor amounts (less than 5%, 4%, 3%, 2%,         1%, 0.5%, 0.25%, or 0.1%) of other ingredients that supplement         sporicidal activity and/or provide a secondary effect (e.g.,         antifogging, soil removal, etc.) that is desirable in view of         the intended end use, and/or inactive additives or adjuvants;     -   “polyacid” means a compound having at least two carboxyl groups         and specifically includes dicarboxylic acids, tricarboxylic         acids, etc.;     -   “pH” means the negative value of the base 10 logarithm of [H+]         as determined by an acceptably reliable measurement method such         as a properly calibrated pH meter, titration curve against a         known standard, or the like;     -   “pK_(a)” means the negative value of the base 10 logarithm of a         particular compound's acid dissociation constant;     -   “E⁰ _(red)” means the standard voltage for a reduction         half-reaction in water at 25° C.;     -   “buffer” means a compound or mixture of compounds having an         ability to maintain the pH of a solution to which it is added         within relatively narrow limits;     -   “buffer precursor” means a compound that, when added to a         mixture containing an acid, results in a buffer;     -   “electrolyte” means a compound that exhibits some dissociation         when added to water;     -   “non-oxidizing electrolyte” means an electrolyte other than one         that can act as an oxidizing agent;     -   “benzalkonium chloride” refers to any compound defined by the         following general formula

where R³ is a C₈-C₁₈ alkyl group, or any mixture of such compounds;

-   -   “effective solute concentration” is a measurement, presented in         units of osmoles per liter, of the colligative property         resulting from the number of moles of molecules (from         nonelectrolyte) or ions (from electrolytes) present in a given         solution;     -   “δp” is the dipolar intermolecular force HSP, with the value for         a solution or mixture of solvents being calculated by

$\begin{matrix} {\delta_{p} = {\sum\limits_{i = 1}^{n}\left( {\delta_{di} \times x_{di}} \right)}} & (I) \end{matrix}$

where δ_(di) h is the energy from dipolar intermolecular force for solvent component i, x_(di) is the percentage of solvent component i relative to the total amount of solvent components, and n is the total number of solvent components;

-   -   “oxyacid” means a mineral acid that contains oxygen;     -   “glycosidase” means an enzyme that assists in hydrolyzing         glycosidic bonds in complex sugars;     -   “substituted” means containing a heteroatom or functionality         (e.g., hydrocarbyl group) that does not interfere with the         intended purpose of the group in question;     -   “microbe” means any type of microorganism including, but not         limited to, bacteria, viruses, fungi, viroids, prions, and the         like;     -   “antimicrobial agent” means a substance having the ability to         cause greater than a 90% (1 log) reduction in the number of one         or more microbes;     -   “active antimicrobial agent” means an antimicrobial agent that         is effective only or primarily during the active parts of the         lifecycle, e.g., cell division, of a microbe;     -   “biocompatible” means presenting no significant, long-term         deleterious effects on or in a mammalian species;     -   “biodegradation” means transformation, via enzymatic, chemical         or physical in vivo processes, of a chemical into smaller         chemical species;     -   “biosorption” means absorption of a material into the body of a         mammalian species;     -   “germicide” means a substance that is lethal to one or more         types of harmful microorganisms;     -   “disinfectant” means a substance that is lethal to one or more         types of bacteria;     -   “high level disinfectant” means a disinfectant that is capable         of killing all bacteria except for small amounts of bacteria in         endospore form;     -   “sterilant” means a substance capable of eliminating at least a         6 log (99.9999%) reduction of all microbes, regardless of form;     -   “contact time” means the amount of time that a composition is         allowed to contact a surface and/or an endospore on such a         surface;     -   “hard surface” means any surface that is non-porous to fluids         and, in most cases, non-deformable; and     -   “healthcare” means involved in or connected with the maintenance         or restoration of the health of the body or mind.

Throughout this document, unless the surrounding text explicitly indicates a contrary intention, all values given in the form of percentages are w/v, i.e., grams of solute per liter of composition. The relevant portion(s) of any specifically referenced patent and/or published patent application are incorporated herein by reference.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The composition will be described first in terms of its properties and components, many of which are widely available and relatively inexpensive, and then in terms of its use.

The solvent component of the composition typically includes a significant amount of water. Relative to its overall volume, a composition often includes up to 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, or even 55% (all v/v). On a per liter basis, a composition often includes from ˜50 to ˜500 mL, commonly from ˜75 to ˜475 mL, more commonly from ˜100 to ˜450 mL, usually from ˜125 to ˜425 mL, typically from ˜150 to ˜400 mL, and most typically 250±50 mL water. The water need not be specially treated (e.g., distilled and/or deionized), although preference certainly can be given to water that does not interfere with the intended antimicrobial effect of the composition.

The solvent component of the composition often includes at least one organic liquid and, in some embodiments, preference is given to those organic liquids with δ_(p) values lower than that of water (δ_(p)≈16.0 MPa^(1/2)). Where an organic liquid or liquids is present in the solvent component, the δ_(p) value of the overall solvent component generally is less than 16.0, no more than 15.6, no more than 15.2, no more than 15.0, no more than 14.6 or no more than 14.0 MPa^(1/2). In some embodiments, the δ_(p) value of the overall solvent component can range from 13.1 to 15.7 MPa^(1/2), from 13.3 to 15.6 MPa^(1/2), from 13.5 to 15.5 MPa^(1/2), and even from 13.7 to 15.4 MPa^(1/2).

The organic liquid(s) often is/are present at concentrations of up to 60%, commonly 5 to 50%, more commonly 10 to 45%, even more commonly 15 to 40%, and typically 20 to 35% (all w/v, based on total volume of solvent component).

The amount of a given organic liquid (or mixture of organic liquids) to be added to water can be calculated using formula (I) if a targeted δ_(p) value is known. Similarly, a projected δ_(p) value can be calculated using formula (I) if the amount of organic liquid(s) and their individual δ_(p) values are known.

The solvent component can consist of, or consist essentially of, only water or only one or more organic liquids, with preference being given to mixtures of species of the same genus of organic liquids, e.g., two ethers or two alcohols rather than one ether and one alcohol. In certain preferred embodiments, the solvent component can consist of, or consist essentially of, water and an organic liquid, preferably one having a δ_(p) value less than 15.5 MPa^(1/2). In yet other embodiments, the solvent component can consist of, or consist essentially of, water and two or more organic liquids, with the resulting solvent component having δ_(p) value that can be calculated using formula (I); again, preference is given to mixtures of species of the same genus of organic liquids, e.g., two ethers or two alcohols rather than one ether and one alcohol.

With respect to organic liquids that can be employed in the solvent component, those which are miscible with one another and/or water are preferred. Non-limiting examples of potentially useful organic liquids include ketones such as acetone, methyl butyl ketone, methyl ethyl ketone and chloroacetone; acetates such as amyl acetate, ethyl acetate and methyl acetate; (meth)acrylates and derivatives such as acrylamide, lauryl methacrylate and acrylonitrile; aryl compounds such as benzene, chlorobenzene, fluorobenzene, toluene, xylene, aniline and phenol; aliphatic alkanes such as pentane, isopentane, hexane, heptane and decane; halogenated alkanes such as chloroform, methylene dichloride, chloroethane and tetrachloroethylene; cyclic alkanes such as cyclopentane and cyclohexane; and polyols such as ethylene glycol, diethylene glycol, propylene glycol, hexylene glycol, and glycerol. When selecting such organic liquids for use in the solvent component of the composition, possible considerations include avoiding those which contain a functional group that will react with either the acid(s)/base(s) or salt(s) employed in the composition and favoring those which possess higher regulatory pre-approval limits.

Preferred organic liquids include ethers and alcohols due to their low tissue toxicity and environmentally friendliness. These can be added at concentrations up to the solubility limit of the other ingredients in the composition.

Ether-based liquids that can be used in the solvent component include those defined by the following general formula

R¹(CH₂)_(x)O—R²—[O(CH₂)_(z)]_(y)Z  (II)

where x is an integer of from 0 to 20 (optionally including, where 2≤x≤20, one or more points of ethylenic unsaturation), y is 0 or 1, z is an integer of from 1 to 4, R² is a C₁-C₆ linear or branched alkylene group, R¹ is a methyl, isopropyl or phenyl group, and Z is a hydroxyl or methoxy group. Non-limiting examples of glycol ethers (formula (II) compounds with Z═OH) that can be used in the solvent component are set forth below in Table 2.

TABLE 2 Representative glycol ethers, with formula (II) variables and δ_(p) values ~δ_(p) R¹ x R² y z (MPa^(1/2)) ethylene glycol monomethyl ether CH₃ 0 (CH₂)₂ 0 — 9.2 ethylene glycol monoethyl ether CH₃ 1 (CH₂)₂ 0 — 9.2 ethylene glycol monopropyl ether CH₃ 2 (CH₂)₂ 0 — 8.2 ethylene glycol monoisopropyl ether (CH₃)₂CH 0 (CH₂)₂ 0 — 8.2 ethylene glycol monobutyl ether CH₃ 3 (CH₂)₂ 0 — 5.1 ethylene glycol monophenyl ether C₆H₅ 0 (CH₂)₂ 0 — 5.7 ethylene glycol monobenzyl ether C₆H₅ 1 (CH₂)₂ 0 — 5.9 diethylene glycol monomethyl ether CH₃ 0 (CH₂)₂ 1 2 7.8 diethylene glycol monoethyl ether (DGME) CH₃ 1 (CH₂)₂ 1 2 9.2 diethylene glycol mono-n-butyl ether CH₃ 3 (CH₂)₂ 1 2 7.0 propylene glycol monobutyl ether CH₃ 3 (CH₂)₃ 0 — 4.5 propylene glycol monoethyl ether CH₃ 1 (CH₂)₃ 0 — 6.5 propylene glycol monoisobutyl ether (CH₃)₂CH 1 (CH₂)₃ 0 — 4.7 propylene glycol monoisopropyl ether (CH₃)₂CH 0 (CH₂)₃ 0 — 6.1 propylene glycol monomethyl ether CH₃ 0 CH₂CH(CH₃) 0 — 6.3 propylene glycol monophenyl ether C₆H₅ 0 CH₂CH(CH₃) 0 — 5.3 propylene glycol monopropyl ether (PGME) CH₃ 2 CH₂CH(CH₃) 0 — 5.6 triethylene glycol monomethyl ether CH₃ 0 (CH₂)₂ 2 2 7.6 triethylene glycol monooleyl ether CH₃ 17* (CH₂)₂ 2 2 3.1 *includes unsaturation at the 9 position

Cyclic and C₁-C₁₆ acyclic (both linear and branched, both saturated and unsaturated) alcohols, optionally including one or more points of ethylenic unsaturation and/or one or more heteroatoms other than the alcohol oxygen such as a halogen atom, an amine nitrogen, and the like, can be included in the solvent component of the composition. Non-limiting examples of representative examples are compiled in the following table.

TABLE 3 Representative alcohols, with δ_(p) values ~δ_(p) (MPa^(1/2)) 2-propenol 10.8 1-butanol 5.7 t-butyl alcohol 5.1 4-chlorobenzyl alcohol 7.5 cyclohexanol 4.1 2-cyclopentenyl alcohol 7.6 1-decanol 10.0 2-decanol 10.0 2,3-dichloropropanol 9.2 2-ethyl-1-butanol 4.3 ethanol 8.8 2-ethyl-hexanol 3.3 isooctyl alcohol 7.3 octanol 3.3 methanol 12.3 oleyl alcohol 2.6 1-pentanol 4.5 2-pentanol 6.4 1-propanol 6.8 2-propanol (IPA) 6.1

For further information on organic liquid-containing solvent components, the interested reader is directed to U.S. Pat. Publ. No. 2016/0073628.

The composition is somewhat acidic. Generally, the composition has a pH of no less than 4.5, and certain embodiments can have a pH of no less than 4.55, 4.60, 4.75, 4.80, 4.85, 4.90, 4.95 or even 5.00. The composition also generally has a pH of no more than 6.75, 6.50, 6.25, or even 6.00. Ranges of pH values employing each of the lower limits in combination with each of the upper limits are envisioned. The composition can have a pH value of 5.5±1.0, 5.6±0.8, 5.7±0.8, 5.75±0.8, 5.8±0.75, and 5.75±0.75.

Acidity can be achieved by adding to the solvent component (or vice versa) one or more acids. Strong (mineral) acids such as HCl, H₂SO₄, H₃PO₄, HNO₃, H₃BO₃, and the like or organic acids, particularly organic polyacids, may be used. Examples of organic acids include monoprotic acids such as formic acid, acetic acid and substituted variants (e.g., hydroxyacetic acid, chloroacetic acid, dichloroacetic acid, phenylacetic acid, and the like), propanoic acid and substituted variants (e.g., lactic acid, pyruvic acid, and the like), any of a variety of benzoic acids (e.g., mandelic acid, chloromandelic acid, salicylic acid, and the like), glucuronic acid, and the like; diprotic acids such as oxalic acid and substituted variants (e.g., oxamic acid), butanedioic acid and substituted variants (e.g., malic acid, aspartic acid, tartaric acid, citramalic acid, and the like), pentanedioic acid and substituted variants (e.g., glutamic acid, 2-ketoglutaric acid, and the like), hexanedioic acid and substituted variants (e.g., mucic acid), butenedioic acid (both cis and trans isomers), iminodiacetic acid, phthalic acid, and the like; triprotic acids such as citric acid, 2-methylpropane-1,2,3-tricarboxylic acid, benzenetricarboxylic acid, nitrilotriacetic acid, and the like; tetraprotic acids such as prehnitic acid, pyromellitic acid, and the like; and even higher degree acids (e.g., penta-, hexa-, heptaprotic, etc.). Where a tri-, tetra-, or higher acid is used, one or more of the carboxyl protons can be replaced by cationic atoms or groups (e.g., alkali metal ions), which can be the same or different.

In certain embodiments, preference can be given to those organic acids which are, or can be made to be, highly soluble in water; acids that include groups that enhance solubility in water (e.g., hydroxyl groups), examples of which include tartaric acid, citric acid, and citramalic acid, can be preferred in some circumstances. In these and/or other embodiments, preference can be given to those organic acids which are biocompatible; many of the organic acids listed above are used in preparing or treating food products, personal care products, and the like.

The composition advantageously need not include an oxidizing acid to have lethal efficacy against endospores. Many oxyacids, such as perchloric, chloric, chlorous, hypochlorous, persulfuric, sulfuric, sulfurous, hyposulfurous, pyrosulfuric, di sulfurous, thiosulfurous, pernitric, nitric, nitrous, hyponitrous, perchromic, chromic, dichromic, permanganic, manganic, perphosphoric, phosphoric, phosphorous, hypophosphorus, periodic, iodic, iodous, etc., are considered to be oxidizing acids. Organic oxidizing acids include, but are not limited to, peracetic acid, peroxalic acid and diperoxalic acid.

If an oxidizing acid is included in the composition, those which have relatively high pK_(a) values (i.e., are not considered to be particularly strong acids) and positive standard potentials (E⁰ _(red)) are preferred. The former permits production of a composition that has a pH value that is not too low, i.e., below ˜1.5, preferably not below ˜1.75, more preferably not below ˜2, and most preferably not below ˜2.2, so that the composition can be used without extreme protective measures by those charged with handling and applying them to surfaces. The latter permits the acid to have sufficient oxidizing capacity to permit overcoming or avoidance of certain endospore defenses such as, for example, oxidation of disulfide linkages and protein polymers on the endospore coat, which allows the outer spore coat to be breached.

Preferred pK_(a) values are greater than ˜1, greater than ˜1.5, greater than ˜2, greater than ˜2.5, greater than ˜3, greater than ˜3.5, greater than ˜4, greater than ˜4.5, greater than ˜5, and even greater than ˜5.5. Acids with lower pK_(a) values can be used if other steps are taken to ensure compliance with required or desired properties of the composition such as pH range (discussed above) and effective solute concentration (discussed below).

Preferred E⁰ _(red) values are those which are at least +0.20 V, at least +0.25 V, at least +0.33 V, at least +0.40 V, at least +0.50 V, at least +0.60 V, at least +0.67 V, at least +0.75 V, at least +0.80 V, at least +0.90 V, at least +1.00 V, at least +1.10 V, at least +1.20 V, or even at least +1.25 V.

Some oxidizing acids are not particularly stable in aqueous solutions. Accordingly, where a composition with an oxidizing acid is desired, preparing the oxidizing acid in vitro can be advantageous. For example, to a solvent component of a composition can be provided acetic acid and hydrogen peroxide which, when contacted, reversibly form peracetic acid.

The amount of any given acid employed can be determined from the target pH of a given composition and the pK_(a) value(s) of the chosen acids in view of the type and amounts of compound(s), if any, utilized to achieve the desired effective solute concentration in the composition. (More discussion of osmolarity and the types of osmolarity-adjusting compounds appears below.)

Also present in the solute component of the composition is an electrolyte oxidizing agent that does not contain any active hydrogen atoms when subjected to the Zerewitinoff determination. Non-limiting examples of potentially useful electrolyte, preferably inorganic, oxidizing agents include compounds which include anions such as manganate, permanganate, peroxochromate, chromate, dichromate, peroxymonosulfate, and the like. (Some of these electrolytes can impact pH, so a composition formulated to have a given pH might require adjustment after addition of the oxidizing agent(s).) Preferred are those compounds having E⁰ _(red) values of at least +1.25 V, preferably at least +1.5 V, more preferably at least +1.75 V, even more preferably at least +2.0 V and most preferably at least +2.25 V.

Electrolyte oxidizing agents generally can be added at up to their individual solubility limits, although the maximum amount generally is on the order of 30 g/L total composition. Exemplary ranges of electrolyte oxidizing agent(s) are ˜2 to ˜25 g/L, ˜3 to ˜21 g/L, ˜4 to ˜18 g/L, ˜5 to ˜16 g/L and ˜6 to ˜14 g/L. Exemplary amounts of electrolyte oxidizing agent(s) are 17.5±12 g/L, 15±9 g/L, 12.5±6 g/L and 10±3 g/L. Utilizing lower amounts of electrolyte oxidizing agents generally results in an increase in biocompatibility of the composition.

Once the acid(s) and oxidizing agent(s) are added to a solvent component that contains water (or vice versa), each at least partially dissociates.

In certain embodiments, the acid(s) and oxidizing agent(s) can provide a relatively high effective solute concentration to the composition. Often, efficacy increases as effective solute concentration (osmolarity) increases. The presence of an abundance of solutes ensures that a sufficient amount are present to induce a high osmotic pressure across the cortical membrane, leading to lysis. This efficacy is independent of the particular identity or nature of individual compounds of the solute component, although smaller molecules are generally more effective than larger molecules due to solvent capacity (i.e., the ability to (typically) include more small molecules in a given amount of solvent component than an equimolar amount of larger molecules) and ease of transport across cortical membranes.

Any of a number of solutes can be used to increase the composition osmolarity, which increases the differential osmotic pressure across the bacterial cell wall membrane.

One approach to achieving increased osmolarity of the composition is by adding large amounts of non-oxidizing electrolytes, particularly ionic compounds (salts); see, e.g., U.S. Pat. No. 7,090,882. Like the oxidizing acid and inorganic oxidizing agent, non-oxidizing electrolytes dissociate upon being introduced into a solvent component that includes water.

Where one or more organic acids are used in the composition, one approach to increasing osmolarity without increasing the pH of the composition past a desired target involves inclusion of salt(s) of one or more the acid(s) or the salt(s) of one or more other organic acids. Such compounds, upon dissociation, increase the effective amount of solutes in the composition without greatly impacting the molar concentration of hydronium ions while, simultaneously, providing a buffer system in the composition.

For example, where the composition includes an acid, a fraction up to a many fold excess (e.g., 3× to 10×, at least 5× or even at least 8×) of one or more salts of that (or another) acid also can be included. The identity of the countercation portion of the salt is not believed to be particularly critical, with common examples including ammonium ions and alkali metals. Where a polyacid is used, all or fewer than all of the H atoms of the carboxyl groups can be replaced with cationic atoms or groups, which can be the same or different. For example, mono-, di- and trisodium citrate all constitute potentially useful buffer precursors, whether used in conjunction with citric acid or another organic acid. However, because trisodium citrate has three available basic sites, it has a theoretical buffering capacity up to 50% greater than that of disodium citrate (which has two such sites) and up to 200% greater than that of sodium citrate (which has only one such site).

One preferred acid/buffer precursor combination is acetic acid and an alkali metal salt of acetic acid, e.g., sodium acetate.

Each buffer precursor, when added to a solvent component that contains water (or vice versa), at least partially dissociates.

The solute component of the composition also includes at least one glycosidase. Preferred are those glycosidases which are active toward peptidoglycans. A particularly preferred glycosidase is lysozyme, which effectively hydrolyzes the glycosidic bond between N-acetylmuramic acid and the fourth carbon atom of N-acetylglucosamine.

Surprisingly, the one or more glycosidases can be added at very low amounts and still provide significant enhancement to a composition's efficacy. They generally can be added at up to their individual solubility limits, although the maximum amount generally is on the order of 5 g/L total composition. Exemplary amounts of glycosidase(s) are on the order of at least 0.05, 0.1, 0.15, or 0.2 g/L up to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 g/L, with ranges employing combinations of each of the lower limits with each of the upper limits being contemplated.

Regardless of how achieved, the effective solute concentration of the composition is at least 0.5, 0.75 or 1.0 Osm/L, generally at least 1.25 Osm/L, often at least 1.5 Osm/L, commonly at least 1.75 Osm/L, more commonly at least 2.0 Osm/L, typically at least 2.25 Osm/L, more typically at least 2.5 Osm/L. (As points of comparison, in biological applications, a 0.9% (by wt.) saline solution, which is ˜0.3 Osm/L, typically is considered to be have moderate tonicity, while a 3% (by wt.) saline solution, which is ˜0.9 Osm/L, generally is considered to be hypertonic.) In some embodiments, the composition has an effective solute concentration of at least ˜3.0, at least ˜3.25, at least ˜3.5, at least ˜3.75, or even at least ˜4.0 Osm/L, with the upper limit being defined by the solubility limit of the solutes in the solvent component; in some embodiments, the upper limit of effective solute concentration can range as high as 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8 or even ˜9 Osm/L. Effective solute concentration ranges involving combinations of any of the lower and upper limits set forth in this paragraph also are envisioned. Surprisingly, the effective solute concentrations of compositions according to the present invention, which are intended to be effective against (i.e., lethal to) endospores, need not be significantly higher than those described in U.S. Pat. Nos. 8,940,792 and 9,314,017 as well as U.S. Pat. Publ. Nos. 2010/0086576, 2013/0272922, 2013/0079407 and 2016/0073628, all of which are directed generally against planktonic bacteria and biofilms.

Unlike many of the compositions described in the documents listed in the preceding paragraph, the present composition does not require inclusion of surfactant in the solute component, although certain preferred embodiments do include one or more wetting agents which include, but are not limited to, surfactants.

Essentially any material having surface active properties in water can be employed, regardless of whether water is present in the solvent component of the composition, although those surface active agents that bear some type of ionic charge are expected to have enhanced antimicrobial efficacy because such charges, when brought into contact with a bacteria, are believed to lead to more effective bacterial membrane disruption and, ultimately, to cell leakage and lysis.

Polar surfactants generally are more efficacious than non-polar surfactants, with ionic surfactants being most effective. For polar surfactants, anionic surfactants generally are the most effective, followed by zwitterionic and cationic surfactants, with smaller molecules generally being preferred over larger ones. The size of side-groups attached to the polar head can influence the efficacy of ionic surfactants, with larger size-groups and more side-groups on the polar head potentially decreasing the efficacy of surfactants.

Potentially useful anionic surfactants include, but are not limited to, ammonium lauryl sulfate, dioctyl sodium sulfosuccinate, perflourobutanesulfonic acid, perfloruononanoic acid, perfluorooctanesulfonic acid, perfluorooctanoic acid, potassium laurylsulfate, sodium dodeylbenzenesulfonate, ladium laureth sulfate, sodium lauroyl sarcosinate, sodium myreth sulfate, sodium myreth sulfate, sodium pareth sulfate, sodium stearate, sodium chenodeoxycholate, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, sodium dodecyl sulfate (SDS), sodium glycodeoxycholate, sodium lauryl sulfate (SLS), and the alkyl phosphates set forth in U.S. Pat. No. 6,610,314. SLS is a particularly preferred material.

Potentially useful cationic surfactants include, but are not limited to, cetylpyridinium chloride (CPC), cetyl trimethylammonium chloride, benzethonium chloride, 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide, tretadecyltrimethyl ammonium borine, benzalkonium chloride (BK), hexadecylpyridinium chloride monohydrate and hexadecyltrimethylammonium bromide.

Potentially useful nonionic surfactants include, but are not limited to, sodium polyoxyethylene glycol dodecyl ether, N-decanoyl-N-methylglucamine, digitonin, n-dodecyl β-D-maltoside, octyl β-D-glucopyranoside, octylphenol ethoxylate, polyoxyethylene (8) isooctyl phenyl ether, polyoxyethylene sorbitan monolaurate, and polyoxyethylene (20) sorbitan cholamidopropyl) dimethylammonio]-2-hydroxy-1-propane sulfonate, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate, 3-(decyldimethylammonio) propanesulfonate inner salt, and N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate.

Potentially useful zwitterionic surfactants include sulfonates (e.g. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), sultaines (e.g. cocamidopropyl hydroxysultaine), betaines (e.g. cocamidopropyl betaine), and phosphates (e.g. lecithin).

For other potentially useful materials, the interested reader is directed to any of a variety of other sources including, for example, U.S. Pat. Nos. 4,107,328, 6,953,772, 7,959,943, and 8,940,792.

The amount(s) of wetting agent(s) to be added to the composition is limited to some extent by the target effective solute concentration and compatibility with other subcomponents of the solute component of the composition. The total amount of surfactant present in the composition can range from at least 0.1%, from at least 0.12%, from at least 0.15%, from at least 0.17% or from at least 0.18% up to 5%, commonly up to 4%, more commonly up to 3%, and typically up to 2.5%. At times, even lower maximum amounts of certain types of surfactants that can be present in a composition for a particular end use (without specific testing, review and approval) are mandated by governmental regulations.

If more than one type of surfactant is employed, the majority preferably is an ionic surfactant, with the ratio of ionic-to-nonionic surfactant generally ranging from ˜2:1 to ˜10:1, commonly from ˜5:2 to ˜15:2, and typically from ˜3:1 to ˜7:1. Additionally, as is known in the art, a composition should not include surfactant types that are incompatible, e.g., anionic with cationic or zwitterionic with either anionic or cationic.

The antimicrobial composition can include a variety of additives and adjuvants to make it more amenable for use in a particular end-use application with negatively affecting its efficacy in a substantial manner. Examples include, but are not limited to, emollients, fungicides, fragrances, pigments, dyes, defoamers, foaming agents, flavors, abrasives, bleaching agents, preservatives (e.g., antioxidants) and the like. A comprehensive listing of additives approved by the U.S. Food and Drug Administration is available as a zipped text file at http://www.fda.gov/Drugs/InformationOnDrugs/ucm113978.htm (link active as of filing date of this application).

The composition does not require the inclusion of an active antimicrobial agent (defined above) to be efficacious, but such materials optionally can be included. Non-limiting examples of potentially useful active antimicrobial additives include C₂-C₈ alcohols (other than or in addition to any used as an organic liquid of the solvent component) such as ethanol, n-propanol, and the like; aldehydes such as gluteraldehyde, formaldehyde, and o-phthalaldehyde; formaldehyde-generating compounds such as noxythiolin, tauroline, hexamine, urea formaldehydes, imidazolone derivatives, and the like; anilides, particularly triclocarban; biguanides such as chlorhexidine and alexidine, as well as polymeric forms such as poly(hexamethylene biguanide); dicarboximidamides (e.g., substituted or unsubstituted propamidine) and their isethionate salts; halogen atom-containing or releasing compounds such as bleach, ClO₂, dichloroisocyanurate salts, tosylchloramide, iodine (and iodophors), and the like; silver and silver compounds such as silver acetate, silver sulfadiazine, and silver nitrate; phenols, bis-phenols and halophenols (including hexachlorophene and phenoxyphenols such as triclosan); and quaternary ammonium compounds.

The following tables provide ingredient lists for exemplary compositions according to the present invention which are free of inorganic oxidizing acids, with amounts being provided in grams and with distilled water being added to bring the ingredients up to a volume of 1 L.

TABLE 4 Formulations for exemplary compositions Formulation 1 Formulation 2 salt of organic acid  8-32 12-24 organic acid 125-200 140-180 ionic surfactant  5-30 15-25 nonionic surfactant 0-5 1-3 glycosidase 0.05-1   0.1-0.5 inorganic oxidizing agent  4-20  6-12 organic liquid 175-450 225-375

Various embodiments of the composition have been provided by way of example and not limitation. As evident from the foregoing table, general preferences regarding features, ranges, numerical limitations and embodiments are, to the extent feasible and as long as not interfering or incompatible, envisioned as being capable of being combined with other such generally preferred features, ranges, numerical limitations and embodiments.

A composition according to the present invention is intended to be, and in practice is, aggressively antimicrobial. Its intended usages are in connection with inanimate objects such as, in particular, hard surfaces, particularly those commonly found in healthcare facilities.

The composition can be applied to inanimate objects, particularly hard surfaces, in a variety of ways including pouring, spraying or misting, via a distribution device (e.g., mop, rag, brush, textile wipe, etc.), and the like.

Alternatively, certain objects are amenable to being immersed in a composition. This is particularly true of medical equipment designed for use with multiple patients such as, for example, dialysis equipment, any of a variety of endoscopes, duodenoscopes, etc., endoscopic accessories such as graspers, scissors and the like, manual instruments such as clamps and forceps, laparoscopic surgical accessories, orthopedic and spinal surgery hardware such as clamps and jigs, and the like. Because the composition of the present invention has a more moderate [H⁺] than treatments such as peracetic acid and bleach, it can achieve disinfection, high level disinfection or even sterilization without negative effects such as, e.g., polymeric degradation, metal corrosion, glass or plastic etching, and the like.

Once applied to a surface or object, the various ingredients of the composition act on any endospores present and avoid or break down their various defenses. The contact time necessary for a composition to treat endospores (i.e., ensure that they cannot return to a vegetative state) can vary widely depending on the particular composition and its intended end use.

A composition intended for use as an immersion bath for medical devices can achieve at least a 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 log reduction after a contact time of no more than 14,400 seconds, no more than 10,800 seconds, no more than 7200 seconds, no more than 5400 seconds, no more than 3600 seconds, no more than 3000 seconds, no more than 2700 seconds, no more than 2400 seconds, no more than 2100 seconds, no more than 1800 seconds, no more than 1500 seconds, no more than 1440 seconds, no more than 1380 seconds, no more than 1320 seconds, no more than 1260 seconds, or even no more than 1200 seconds. When tested in accordance with a variant of AOAC Official Method 966.04, Method II (substituting C. diff spores for B. subtilis, using stainless steel cylinders rather than porcelain, performing all incubations under anaerobic conditions, and using Brain-Heart infusion agar), certain embodiments can achieve a passing score after contact times of no more than 900, 600 or even 300 seconds.

A composition intended to be applied to hard surfaces, particularly surfaces in a healthcare facility, is expected to be able to achieve at least a 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8 or 5.0 log reduction after a contact time of more than 1200 seconds, no more than 1050 seconds, no more than 900 seconds, no more than 840 seconds, no more than 780 seconds, no more than 720 seconds, no more than 660 seconds, or even no more than 600 seconds. When tested in accordance with ASTM E2197-11, embodiments of the composition are expected to achieve at least a 3 log reduction after a contact time of 600 seconds.

After the composition has been allowed to contact a given object or surface for an appropriate time (in view of factors such as expected bacterial load, type of bacteria potentially present, importance of the object/surface, any pre-cleaning that might have been undertaken, the presence and amount of soil load, and the like), it can be left to evaporate or, preferably, rinsed away with water or a dilute saline solution.

The following non-limiting, illustrative examples provide detailed conditions and materials that can be useful in the practice of the present invention. Throughout those examples, any reference to room temperature refers to ˜22° C.

EXAMPLES Examples 1-2: Control and Comparative

A 0.01 M phosphate-buffered saline solution was prepared by adding one phosphate-buffered saline tablet (Sigma-Aldrich Corp.; St. Louis, Mo.) to 200 mL distilled water. This solution was covered and stored at room temperature.

VIRASEPT™ disinfectant (Ecolab USA; St. Paul, Minn.) was kept at room temperature in its as-purchased form and used without modification.

Examples 3-6

Four compositions were prepared by adding to 100 mL glass vessels each of the following ingredients (amounts in grams):

buffer precursor (sodium acetate) 1.49 acid (acetic acid) 16.63 organic liquid (DGME) 30.0 anionic surfactant (SDS) 1.75 nonionic surfactant (polyoxyethylene (20) 0.2 sorbitan monooleate) The compositions also included electrolyte oxidizing agent (KHSO₅), glycosidase (lysozyme), and sodium hydroxide, with the varying amounts (in grams) tabulated below. Sodium hydroxide solution (50% w/v in water) was dropwise added to upwardly adjust the pH of the compositions to the values shown in the following table.

TABLE 5 Ex. 3 Ex. 4 Ex. 5 Ex. 6 KHSO₅ 0.8 0.8 1.0 1.0 lysozyme 0.015 0.03 0.03 0.03 NaOH 3.75 3.75 3.75 8.33 pH 6 6 6 5

After the aforementioned pH adjustment, sufficient distilled water was added to bring each composition to 100 mL.

The comparative and inventive compositions were allowed to contact C. difficile endospores (ATCC 43598) for ˜10 minutes using the procedure of ASTM E2197-11, with their efficacy being reported in Table 6 below as log reductions in the number of viable endospores after the noted contact time.

Testing also was performed on steel cylinders according to a variant of AOAC Official Method 966.04, Method II, a brief description of which follows:

-   -   To a 20 mL beaker containing 10 mL of the type of C. diff         suspension described in the U.S. Environmental Protection         Agency's “Standard Operating Procedure for Production of Spores         of Clostridium difficile for Use in the Efficacy Evaluation of         Antimicrobial Agents” (SOP #MB-28-00), a sufficient number of         steel cylinders were added so as to provide 10 per test to be         run. Cylinders were permitted to sit in this suspension for         10-15 minutes before being removed, placed in a petri dish and         dried under a vacuum for 24 hours.     -   Each composition was provided in duplicate (i.e., two vessels of         each composition), with a maximum of 5 inoculated cylinders         placed in a vessel containing 10 mL of tested composition.         Compositions were tested at both 30 and 60 minutes, with each         time duration involving duplicate tested compositions.     -   After being exposed to a tested composition for the         predetermined time, each cylinder was transferred sequentially         to a primary subculture tube containing 10 mL room temperature         pre-reduced Dey/Engley neutralizing broth and, within one hour         of all tested cylinders being transferred to a primary tube, a         secondary subculture tube containing 10 mL reinforced         clostridial medium (Becton Dickinson and Co.; Franklin Lakes,         N.J.—38 g in 1 L distilled water, autoclaved at 121° C. for ˜15         minutes, then pre-reduced in an anaerobic chamber for ˜24         hours). Each type of subculture tube was incubated for 21 days         at 36°±1° C.

This same procedure also was performed with 2-loop coils of black, braided, polyester surgical sutures (USP size, metric size 6.0) instead of steel cylinders. A recommended method of preparing such coils is described in the aforementioned AOAC test.

Results are recorded as positive or negative growth which, in the following table, is presented in the form of x/y where x is the number of cylinders with positive growth and y is the total number of cylinders tested.

TABLE 6 Test results ASTM E2197-11 AOAC (steel) AOAC (suture) (log reduction) 30 min. 60 min. 30 min. 60 min. Ex. 1 (control) — 19/20  10/10  20/20  10/10  Ex. 2 3.87 1/20 0/10 6/20 3/10 (comparative) Ex. 3 3.75 0/20 0/10 0/20 0/10 Ex. 4 3.56 (a) (a) (a) (a) Ex. 5 3.19 9/20 5/10 0/20 1/10 Ex. 6 3.66 0/10 0/10 0/20 0/10 (a) not tested due to results of preliminary evaluation of Ex. 3

Each of the exemplary compositions had at least some efficacy against C. difficile endospores, with the best results being achieved with Example 3 (pH≈6, lowest amounts of electrolyte oxidizing agent and glycosidase) and Example 6 (pH≈5, slightly higher amounts of electrolyte oxidizing agent and glycosidase). The composition of Example 4 is expected to provide results at least as good as those of Example 3.

The efficacy results of these compositions compare favorably with those of the comparative composition (Example 2), a commercial disinfectant that contains H₂O₂, peroxyacetic acid and octanoic acid and has a [H⁺] two to three orders of magnitude greater, i.e., pH of 3.0±0.5 according to its material safety data sheet.

Thus, despite being significantly less acidic than the comparative, the compositions of Examples 3-6 had efficacies roughly equivalent to that of a commercial disinfectant. 

1-14. (canceled)
 15. A composition useful for treating surfaces susceptible to carry endospores, comprising: a) a solvent component that comprises water and one or more organic liquids, said solvent component exhibiting a δ_(p) value of from 13.1 to 15.7 MPa^(1/2), and b) a solute component that comprises (1) a buffer provided by combining at least one carboxylic acid and conjugate base of at least one carboxylic acid, (2) dissociation product of from 4 to 18 g/L of one or more electrolyte oxidizing agents, (3) from 0.05 to 1 g/L of a glycosidase, and (4) from 5 to 30 g/L of an ionic surfactant, said composition having a pH of from about 4.75 to about 6.5 and an effective solute concentration of at least 0.5 Osm/L.
 16. The composition of claim 15 wherein said ionic surfactant is an anionic surfactant.
 17. The composition of claim 15 wherein said one or more organic liquids comprises at least one glycol ether.
 18. The composition of claim 15 wherein said solute component further comprises nonionic surfactant.
 19. The composition of claim 18 wherein said ionic surfactant is an anionic surfactant.
 20. The composition of claim 18 wherein said solute component further comprises dissociation products of one or more non-oxidizing electrolytes.
 21. The composition of claim 16 wherein said solute component further comprises dissociation products of one or more non-oxidizing electrolytes.
 22. The composition of claim 15 wherein said solute component further comprises dissociation products of one or more non-oxidizing electrolytes.
 23. The composition of claim 22 wherein said composition is free of oxidizing acids.
 24. The composition of claim 15 wherein said composition is free of oxidizing acids.
 25. The composition of claim 15 wherein at least one of said one or more electrolyte oxidizing agents comprises manganate, permanganate, peroxochromate, chromate, dichromate, or peroxymonosulfate anion.
 26. The composition of claim 15 wherein at least one of said one or more electrolyte oxidizing agents has an E⁰ _(red) value of at least +1.5 V.
 27. A composition useful for treating surfaces susceptible to carry endospores, said composition being free of oxidizing acids and consisting of: a) a solvent component that comprises water and one or more organic liquids, said solvent component exhibiting a δ_(p) value of from 13.1 to 15.7 MPa^(1/2), and b) a solute component that comprises (1) a buffer provided by combining at least one carboxylic acid and conjugate base of at least one carboxylic acid, (2) dissociation product of from 4 to 18 g/L of one or more electrolyte oxidizing agents, (3) from 0.05 to 1 g/L of a glycosidase, (4) from 5 to 30 g/L of an anionic surfactant, and (5) a nonionic surfactant, said composition having a pH of from about 4.75 to about 6.5 and an effective solute concentration of at least 0.5 Osm/L.
 28. The composition of claim 27 in which said effective solute concentration is at least 1.5 Osm/L.
 29. The composition of claim 27 wherein said buffer comprises acetic acid and an alkali metal salt of acetic acid.
 30. The composition of claim 27 wherein said buffer is provided by combining acetic acid and an alkali metal salt of acetic acid.
 31. The composition of claim 27 wherein said solute component further comprises dissociation products of one or more non-oxidizing electrolytes.
 32. The composition of claim 27 wherein said solute component comprises from 7 to 13 g/L of said one or more electrolyte oxidizing agents.
 33. The composition of claim 32 wherein at least one of said one or more electrolyte oxidizing agents comprises manganate, permanganate, peroxochromate, chromate, dichromate, or peroxymonosulfate anion.
 34. The composition of claim 27 wherein at least one of said one or more electrolyte oxidizing agents comprises manganate, permanganate, peroxochromate, chromate, dichromate, or peroxymonosulfate anion. 